Hormone dependent breast cancers initially respond to endocrine therapy with tumor regression but tumor recurrences are invariable. An understanding of the mechanisms mediating relapse would allow development of strategies to counteract this process. We have provided evidence that tumor cells adapt during treatment by becoming hypersensitive to estradiol, a process which mediates tumor re-growth. We wish to determine precisely what steps induce this adaptive process. Preliminary data suggest a key role for cell cycle events distal to estrogen receptor function. Our hypothesis is that an adaptive process occurs in which growth factor and estradiol mediated events cooperatively interact at a "point of intersection" in the cell cycle to mediate hypersensitivity. At this "intersection point," c-myc, a direct estrogen-mediated early response gene, and products of the growth factor stimulated RAS pathway cooperate to regulate key cell cycle proteins. We postulate that this interaction results in suppression of the cyclin inhibitor p-27, bypass of the need for phosphorylation of the Rb protein, enhanced activation of E2F transcription factors, and an increase in cyclin E kinase activity. To test this hypothesis, we will pursue four specific aims using as a model, breast cancer cells with adaptive hypersensitivity. Specific Aim 1 will evaluate the role of E2F transcription factors and the cyclins in the process of adaptive hypersensitivity. Specific Aim 2 will seek to demonstrate down regulation of cyclin inhibitors and resultant effects on E2F levels and cell proliferation. We postulate that adaptation to estrogen deprivation involves down regulation of inhibitors of cyclin dependent kinase activation. We will measure the levels of the two classes of inhibitors, the INK 4 a-d group as well as the Cip-1/Waf-1/Kip1/Kip2 class (p-2l, p-27, p-57). To directly assess cause and effect relationships, we will use stable transfectants to determine if p-27 activation decreases sensitivity to estradiol in LTED deprived cells. Specific Aim 3 will evaluate the role of growth factor pathways on E2F production. We will use strategies both for induction of hypersensitivity in wild type cells and for reversion of hypersensitive cells back to normal sensitivity. Inhibitors of MAP kinase and RAS plasma membrane binding will be used to interdict the effects of activated RAS in hypersensitive cells and to cause reversion of hypersensitivity. induction of hypersensitivity in wild type cells will involve TGF alpha administration as well as use of tetracycline regulatable, stable transfection constructs to cause over-expression of MAP kinase. Specific Aim 4 will determine whether LTED deprivation induces antiapoptotic regulatory mechanisms. These studies are expected to identify appropriate targets which could then be used in patients to revert hypersensitive cells back to normal and thereby enhance the duration of hormonal therapy in women with breast cancer.